Project Summary/Abstract Methanobactins (Mbns) are ribosomally-synthesized and post-translationally modified peptide natural products produced by methanotrophic (methane-oxidizing) bacteria to obtain copper from the environment; copper is necessary for their primary metabolic enzyme, copper- dependent particulate methane monooxygenase (pMMO). Previously characterized Mbns contain a peptidic backbone with two heterocycle/thioamide pairs that provide two nitrogen and two sulfur ligands for Cu(I). These Mbns bind copper with high affinity and specificity, and are under investigation as drug candidates for human diseases of copper metabolism. Recent bioinformatics studies have revealed that there are new types of Mbns that are predicted to differ in structure and chemical properties from previously characterized Mbns. Moreover, several uncharacterized enzymes that are expected to be involved in the biosynthesis of these new Mbns are not well understood. In this project, uncharacterized Mbns with predicted novel structures will be explored, and the enzymes responsible for their biosynthesis will be studied by both in vivo and in vitro approaches. This research will provide significant insights into the diversity of Mbn structures and functions, will elucidate new enzymatic chemistries, and will impact development of Mbns and Mbn-like molecules as therapeutics. A multifaceted training program has been planned. Training in a variety of biochemical and genetic as well as spectroscopic/spectrometric techniques needed to isolate and characterize Mbns and their biosynthetic enzymes will be provided in the Rosenzweig laboratory. In addition, formal training opportunities outside the laboratory, such as programs at the Life Sciences Collaborative Access Team (LS-CAT) beamlines at the Advanced Photon Source (APS) at Argonne National Laboratory and at the Penn State University Bioinorganic Training Workshop, both of which are closely related to the proposed research, will be available. The Department of Molecular Biosciences at Northwestern University is equipped with all the equipment and resources necessary to conduct this research. In particular, collaborations with the Hoffman laboratory for electron paramagnetic resonance (EPR) spectroscopy, Dr. Zhang at the Integrated Molecular Structure Education and Research Center (IMSERC) for a suite of NMR spectroscopic techniques, and Kelleher group for mass spectrometry will facilitate carrying out this research. These training opportunities combined with the collaborative and scientific environment at Northwestern University will ensure the success of the proposed research and will provide excellent preparation for an independent, interdisciplinary research career.